Cardiovascular disease (CVD) is a general term used to classify numerous conditions that affect the heart, heart valves, blood, and vasculature of the body. Cardiovascular diseases include coronary artery disease, angina pectoris, myocardial infarction, atherosclerosis, congestive heart failure, hypertension, cerebrovascular disease, stroke, transient ischemic attacks, cardiomyopathy, arrhythmias, aortic stenosis, and aneurysm. Signs and symptoms of cardiovascular disease include chest, neck, or arm pain, palpitations (irregular heart beat), dyspnea (shortness of breath), syncope (fainting), fatigue, cyanosis (bluish coloration of the lips and nails), and claudication (leg pain).
Cardiovascular disease remains the number one killer of people in the United States today. The diagnosis of CVD is made by assessing a patient's clinical symptoms, by running laboratory tests to determine levels of certain enzymes, as well as by coronary angiography, electrocardiogram, and an exercise stress test (treadmill).
There are many risk factors that may contribute to the development of CVD. Certain of these risk factors are modifiable. These include cigarette smoking, high LDL cholesterol, low HDL cholesterol, diabetes, hypertension, and physical inactivity. Other contributing risk factors include obesity, diet, and alcohol consumption. Some risk factors are not capable of being modified and these include age, sex, race, and family history.
The optimal treatment for CVD is prevention and modification of risk factors. If the disease has progressed beyond prevention and modification, surgical intervention including percutaneous transluminal coronary angioplasty (PTCA), coronary bypass, and coronary stents may be performed and or implanted.
While the risk factors for CVD are used by physicians in risk prediction matrices in an attempt to target those individuals who are at highest risk for development of CVD, thereby allowing these individuals to modify their lifestyle to lower their risk profile to the extent possible, these algorithms are still limited in their predictability. Accordingly, there is a need for expanding these algorithms to take into account other factors that should be included in a patient's risk profile for development of CVD.
It is generally recognized that many disease processes are associated with the presence of elevated levels of oxidative stress induced compounds, such as free radicals and reactive oxygen species (ROS) and reactive nitrogen species (RNS). These include superoxide, hydrogen peroxide, singlet oxygen, peroxynitrite, hydroxyl radicals, hypochlorous acid (and other hypohalous acids) and nitric oxide.
For example, in the eye, cataract, macular degeneration and degenerative retinal damage are attributed to ROS. Other organs and their ROS-related diseases include: lung cancer induced by tobacco combustion products and asbestos; accelerated aging and its manifestations, including skin damage and scleroderma; atherosclerosis; ischemia and reperfusion injury, diseases of the nervous system such as Parkinson disease, Alzheimer disease, muscular dystrophy, multiple sclerosis; lung diseases including emphysema and bronchopulmonary dysphasia; iron overload diseases such as hemochromatosis and thalassemia; pancreatitis; diabetes; renal diseases including autoimmune nephrotic syndrome and heavy metal-induced nephrotoxicity; and radiation injuries. Diseases of aging and chronic emotional stress also appear to be associated with a drop in glutathione levels, which allows ROS to remain active.
However, while there has been an association of these disease states with high levels of oxidative stress induced compounds, the reliance of these compounds for use as a marker of risk for development of these diseases has not been demonstrated. On the other hand, there is current evidence in animal studies that oxidation of LDL occurs in vivo, and the results suggest that this may lead to the formation and build up of atherosclerotic plaques.
A wealth of evidence suggests that LDL must be oxidatively modified to damage the artery wall (Heinecke, (1998) Atheroscler. 141, 1-15). One pathway for LDL oxidation in humans has been described (Daugherty et al. (1994) Journal of Clinical Investigation 94, 437-444). It involves hypochlorous acid and other reactive intermediates generated by myeloperoxidase, a heme protein secreted by phagocytes. High concentrations of enzymatically active myeloperoxidase have been found in human vascular lesions (Sugiyama et al., (2001) Am J Pathol 158, 879-891.), and the enzyme's characteristic protein and lipid oxidation products have been detected in LDL isolated from atherosclerotic tissue (Hazen, et al., (1997) J. Clin. Invest. 99, 2075-2081; Heller et al., (2000) J. Biol Chem 275, 9957-9962; Leeuwenburgh et al., (1997) J. Biol. Chem. 272, 3520-3526).
Another oxidative pathway involves nitric oxide (nitrogen monoxide; NO), which is generated by vascular wall cells (Moncada, et al., (1991) Pharmacological Reviews 43, 109-142). NO is a relatively stable free radical that is unable to oxidize LDL directly under physiological conditions (Beckman, et al. (1996) Am J Physiol 271, C1424-1437; Ischiropoulos, (2003) Biochem Biophys Res Commun 305, 776-783). However, it reacts rapidly with superoxide to form peroxynitrite (ONOO−) (Beckman et al., (1990) Proceedings of the National Academy of Sciences of the United States of America 87, 1620-1624), a reactive nitrogen species that promotes peroxidation of the lipid moiety of LDL in vitro (Graham et al., (1993) FEBS Letters 330, 181-185). Proteins also appear vulnerable to ONOO− because the oxidant reacts in vitro with tyrosine residues to yield the stable product 3-nitrotyrosine (Beckman, et al, (1994) Methods in Enzymology 233, 229-240). LDL isolated from human atherosclerotic lesions contains much higher levels of 3-nitrotyrosine than does circulating LDL, as monitored by isotope dilution gas chromatography-mass spectrometry (GC/MS), a sensitive and specific method (Leeuwenburgh et al., (1997) Journal of Biological Chemistry 272, 1433-1436). These observations indicate that reactive nitrogen species oxidize LDL in the human artery wall.
Cultured endothelial cells, macrophages, and smooth muscle cells, all components of the atherosclerotic lesion, generate superoxide anion. Moreover, elevated levels of nitrated plasma proteins associate with an increased risk of coronary artery disease, suggesting that oxidants derived from NO modify circulating proteins or proteins that find their way into the bloodstream (Shishehbor et al., (2003) Jama 289, 1675-1680). Fibrinogen is one target for nitration in plasma. Also, exposing fibrinogen to nitrating oxidants in vitro accelerates clot formation (Vadseth et al., (2004) J Biol Chem 279, 8820-8826).
NO can also autoxidize to nitrite (NO2−), and plasma levels of NO2− rise markedly during acute and chronic inflammation (Farrell et al., (1992) Ann Rheum Dis 51, 1219-1222). Because NO2− is a substrate for myeloperoxidase and other peroxidases, it may also be used to nitrate tyrosine in vivo (Klebanoff, (1993) Free Radio Biol Med 14, 351-360; Chance, (1952) Arch Biochem Biophys 41, 425-431). Indeed, myeloperoxidase uses hydrogen peroxide (H2O2) and NO2− to generate reactive nitrogen species that nitrate free and protein-bound tyrosine residues and promote lipid peroxidation of LDL in vitro (Eiserich et al., (1996) Journal of Biological Chemistry 271, 19199-19208; Eiserich et al., (1998) Nature 391, 393-397; Byun et al., (1999) FEBS Letters 455, 243-246; Podrez et al., (1999) J Clin Invest 103, 1547-1560. These reactions might be physiologically relevant because tyrosine nitration is markedly impaired in a model of peritoneal inflammation in myeloperoxidase-deficient mice by a reaction pathway that appears to require NO2− or other intermediates derived from NO (Gaut et al., (2002) J Clin Invest 109, 1311-1319). In human atherosclerotic lesions, most cell-associated myeloperoxidase is found in and around macrophages (Daugherty et al., (1994) Journal of Clinical Investigation 94, 437-444). However, the enzyme has also been detected in endothelial cells (Baldus et al., (2001) J Clin Invest 108, 1759-1770), raising the possibility that reactive intermediates produced by peroxidases might generate the epitopes on macrophages and endothelial cells that are recognized by antibodies to 3-nitrotyrosine.
High density lipoprotein (HDL) protects the artery wall against the development of atherosclerosis (reviewed in Miller et al., O.D. 1977. Lancet 1:965-968; Keys, A. 1980. Lancet 2:603-606). This atheroprotective effect is attributed mainly to HDL's ability to mobilize excess cholesterol from arterial macrophages. Cell culture experiments have uncovered several mechanisms that enable components of HDL to remove cellular cholesterol (Oram, et al., 1996. J Lipid Res 37:2473-2491; Rothblat et al., 1999. J Lipid Res 40:781-796). For example, phospholipids in HDL absorb cholesterol that diffuses from the plasma membrane, a passive process facilitated by the interaction of HDL particles with scavenger receptor B1. In contrast, HDL apolipoproteins remove cellular cholesterol and phospholipids by a cholesterol-inducible active transport process mediated by a cell membrane protein called ATP-binding cassette transporter A1 (ABCA1) (5-8).
The most abundant apolipoprotein in HDL is apolipoprotein (apo) A-I, which accounts for ˜70% of HDL's total protein content. Lipid-poor apo A-I promotes efflux of cellular cholesterol and phospholipids exclusively by the ABCA1 pathway (Brooks-Wilson et al., 1999. Nat Genet. 22:336-345; Bodzioch et al., 1999. Nat Genet. 22:347-351; Rust et al., 1999. Nat Genet. 22:352-355; Lawn et al., 1999. J Clin Invest 104:R25-31). This process appears to involve the amphipathic α-helical domains in apo A-I (Oram, J. F. 2003. Arterioscler Thromb Vasc Biol 23:720-727). Studies of synthetic peptides and deletion mutants of apo A-I suggest that the terminal helices of apo A-I penetrate into the phospholipid bilayer of membranes, promoting cooperative interactions between other α-helical segments and lipids to create an apolipoprotein/lipid structure that dissociates from membranes (Gillotte et al, 1999. J Biol Chem 274:2021-2028). This atheroprotective process is inhibited by oxidative damage, which is implicated in the pathogenesis of atherosclerosis (Diaz et al., Jr. 1997. N Engl J Med 337:408-416.).
Myeloperoxidase uses hydrogen peroxide to convert chloride to hypochlorous acid (HOCl), which reacts with tyrosine to form 3-chlorotyrosine (Heinecke, (1998) Atheroscler. 141, 1-15). At plasma concentrations of chloride ion, myeloperoxidase is the only human enzyme known to produce HOCl. Chlorination of the phenolic ring of tyrosine may have physiological relevance because elevated levels of 3-chlorotyrosine and other products characteristic of myeloperoxidase have been detected in LDL isolated from human atherosclerotic lesions (Hazen et al., 1997. J Clin Invest 99:2075-2081; Leeuwenburgh et al., 1997. J Biol Chem 272:3520-3526; Heller et al., 2000. J Biol Chem 275:9957-9962). Moreover, methionine and phenylalanine residues in apo A-I are oxidized by reactive intermediates (Panzenboeck et al., 2000. J Biol Chem 275:19536-19544; Bergt et al., 2000. Biochem J 346 Pt 2:345-354; Garner et al., 1998. J Biol Chem 273:6080-6087), and tyrosine residues are converted to o,o′-dityrosine by tyrosyl radical (Francis et al., 1993. Proc Natl Acad Sci USA 90:6631-6635). HOCl selectively targets tyrosine residues in apo A-I that are suitably juxtaposed to primary amino groups in proteins (Bergt et al., 2004. J Biol Chem 279:7856-7866). This mechanism might enable phagocytes to efficiently damage proteins during inflammation.
There is still a need for diagnostic tests to aid in the characterization of subjects at risk for developing diseases characterized in part by high levels of oxidative stress-induced compounds such as HDL oxidation products, in particular, cardiovascular disease. Furthermore, there is a need to establish whether a specific therapy is having the appropriate effect in individuals suffering from such conditions. Thus, prognostic markers or indicators to monitor the effects of such therapy are also needed.